P329: UNRAVELING THE ROLE OF GATA3 IN EARLY HUMAN T CELL DEVELOPMENT AND EARLY T CELL PRECURSOR ALL (ETP-ALL)
Authors: N. Lambrechts, K. L. Liang, T. Putteman, J. Roels, J. Van Hulle, T. Taghon
Journal: HemaSphere (2022)

Background: ETP-ALL is a subtype of T cell acute lymphoblastic leukemia (T-ALL) and can be identified by a characteristic immunophenotype and gene expression profile. Treatment of relapsed ETP-ALL remains challenging using conventional chemotherapy and little targeted therapies are available. A better understanding in the molecular mechanisms involved in the leukemogenesis and maintenance of ETP-ALL could lead to improved and more targeted treatments. An estimated 9% of ETP-ALL patients carry mutations in GATA3, a transcription factor known to regulate T cell commitment by inducing T lineage genes and inhibiting genes important for other lineages such as NK and B cells. Although these mutations have been described to be loss-of-function mutations, the exact mechanisms through which they contribute to ETP-ALL remain to be elucidated. Aims: We aim to gain insights into the molecular mechanisms through which GATA3 regulates normal human early T cell development and how disruption of GATA3 function, as a result of mutation, can contribute to the emergence of ETP-ALL. Methods: We have studied the effect of the most prevalent GATA3 mutation (c.827G>A, p.R276Q) in ETP-ALL on normal human T cell development using in vitro differentiation cultures. We examined the impact of GATA3 p.R276Q on gene expression, DNA binding and chromatin accessibility through RNA-, CUT&Tag- and ATAC-sequencing respectively, starting from primary CD34+CD1a- early human T cell progenitors isolated from thymus or the ETP-like cell line PER117. Results: While overexpression of wild-type GATA3 in thymic progenitor T cells induces an acceleration of development towards the CD4+CD8ab+ double positive stage, overexpression of the mutant form results in a delay and an accumulation of CD4 immature single positive cells in OP9-DL1 co-cultures. Remarkably, the inhibition of NK cell development, which is an important function of wild-type GATA3, is maintained by GATA3 R276Q. This indicates that GATA3 R276Q is not merely a loss-of-function mutation. RNA-, CUT&Tag- and ATAC sequencing after overexpression in human CD34+CD1a- immature thymocytes or the ETP-ALL-like cell line PER117 revealed that most key regulatory effects are maintained by GATA3 R276Q compared to its wild-type counterpart, including induction of T cell genes and inhibition of stem cell and NK cell genes, binding to its target regions by recognizing GATA motifs and exerting a pioneering function by opening and closing chromatin. Summary/Conclusion: Continuous GATA3-R276Q has a detrimental effect on T cell development in vitro. Since the inhibition on NK cell development is maintained, these findings suggest that this decision is uncoupled from the induction of T cell lineage commitment. Many important effects on RNA expression, DNA binding and chromatin accessibility are maintained by the R276Q mutant compared to wild-type GATA3, pointing out that this mutation not purely acts as a loss-of-function. The exact mechanisms through which GATA3 R276Q contributes to ETP-ALL still remain to be elucidated, but our results show that inhibition of T cell development most likely is one piece of the puzzle that leads to malignant transformation.